*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk ***
evette, i agree that phaser is very nice to use for molrep and quite easy to learn, but does take some trial and error. i would first do a search with your A molecule in Phaser, but search for only 1, not 2 or 3. if it is going to work at all, you should find at least the 1st without much of a problem, for most cases. I would also repeat this for molecule B, and see if you at least get nice Z scores for searching for only 1 molecule, A or B. If multiple copies are present, then (I think) phaser will simply output those solutions also in the .sol file ....but only the top solution will be output as a .pdb file. If mulitple solutions are present in the .sol file, you can use the parameters in the file to create coordinates for those additional solutions using PDBSET within CCP4. Then combine the files into a single coordinates file. I must admit though, since you already have existing structures for both A and B, I can't explain why EPMR didn't work (unless major conformational changes occur), if molrep is going to work at all. yet, i am not familiar with EPMR much at all. i suspect, given the size or your proteins and your resolution, you probably get nice xtals, right? is it possible for you to harvest/wash/wash/wash your xtals and then run a gel to give you an idea about the possible ratios of A and B? At the very least, you could confirm whether or not you actually have both A and B present in the complex. You could also run a native gel of the xtals and then compare that to your protein solution, and see if the complex in your crystal represents the complex in solution, A:B. HOpe some of this helps. best of luck. if you have more questions about phaser, feel free to email anytime. cheers, nick -----Original Message----- From: "Radisky, Evette S. Ph.D." <[EMAIL PROTECTED]> To: <[email protected]> Date: Mon, 6 Nov 2006 11:50:59 -0600 Subject: [ccp4bb]: Best bet for a molecular replacement problem Dear all, In the past I have only ever used epmr for molecular replacement, and have never encountered any tough cases where that didn't work. I have never before had to deal with multiple copies in the asymmetric unit. I am now struggling to solve the structure of a complex between A (a 24 kDa protein) and B (a 6.5 kDa protein). My native data set extends to 1.4A resolution; space group is C222 or C2221. For models, I have a 1.7A structure of A alone, and a 1.6A structure of B alone. Unit cell analysis suggests that there should be 2 copies of the A-B complex in the asymmetric unit. (Although the affinity between A and B is not that great, and it is possible that I have crystallized A alone or B alone; eg, there could be 3 copies of A and no B in the asymmetric unit.) So far, epmr has failed me, and I spent the weekend reading up on Phaser documentation and trying it out, still with no success. For those programs, I was trying both possible space groups, and trying to search with both models as pieces of the complete model, as well as trying to search with my A model alone, for 2 or 3 copies. I am now reading up on Molrep and have started an auto MR run; I guess I will try the DYAD option next (as mentioned in a recent posting) if the first run doesn't give a solution. A couple of questions about this: in using the DYAD=D option for Molrep, how should I choose appropriate choices for "number of peaks" variables NP, NPT, and NPTD? And in setting the variables refering to the model, if I am searching for A alone, is my "fraction completeness of model" 0.8 (80% of one A-B complex) or 0.4 (40% of the contents of the asymmetric unit)? I would also welcome any input about these or other favorite programs and approaches for situations like this one. Also, I find that program documentation often doesn't give a lot of guidance for selection of best options and variables, so any hints on those that may be critical for my situation would also help. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-2857 (lab) ________________________________________ Nicholas Noinaj University of Kentucky College of Medicine Department of Molecular and Cellular Biochemistry The Center for Structural Biology Biomedical Biological Sciences Research Building, Rm 236 741 S. Limestone Lexington, Ky 40536 Lab: 859-323-8183 Cell: 859-893-4789 Home: 859-228-0978 [EMAIL PROTECTED] noinaj.com
